Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of infecting a wide variety of animals, plants, and insects (Rahme et al., Science 268:1899-1902 (1995)). P. aeruginosa is classified as an opportunistic pathogen because while it rarely causes disease in healthy people, it does infect people with impaired host defense systems that include cystic fibrosis (CF), cancer, AIDS, diabetes, deep burns, and wounds (Van Delden et al., Emerg. Infect. Dis. 4:551-560 (1998)). During infection, P. aeruginosa produces numerous virulence factors that cause tissue damage. In the case of CF, P. aeruginosa causes chronic, fatal lung infections in over 90% of patients (Hoiby, Acta. Path. Microbiol. Scand. Sect. B 82:551 (1974); Mahenthiralingam et al., Infect Immun. 62:596-605 (1994); Smith et al., Cell 85:229-236 (1996)). Although several anti-pseudomonal antibiotics, such as β-lactams, aminoglycosides and quinolones, are used in clinical drug regimens (Kovacs et al., Infect. Med. 15:467-472 (1998)), P. aeruginosa frequently develops resistance to these treatments (Oliver et al., Science 288:1251-1253 (2000)). Compounding the difficulty in eliminating the infection is the intrinsic ability of P. aeruginosa to develop a biofilm (Costerton et al., Science 284:1318-1322 (1999)). A biofilm is a complex, polysaccharide-laden microniche that protects the bacteria from both antibiotics and the host's humoral and cell-mediated responses.
Regulation of virulence factor production and biofilm development in P. aeruginosa is controlled by a sophisticated inter-cellular signaling mechanism responding to cell population density, known as quorum sensing (QS) (de Kievit et al., Sci. Med. November/December:42-50 (1999); Passador et al., Science 260:1127-1130 (1993); Passador et al., J. Bacteriol. 178:5995-6000 (1996); Hastings et al., J. Bacteriol. 181:2667-2668 (1999); Pesci et al., Proc. Natl Acad. Sci. USA 96:11229-11234 (1999); Parsek et al., Proc. Natl Acad. Sci. USA 97:8789-8793 (2000); Whiteley et al., Proc. Natl Acad. Sci. USA 96:13904-13909 (1999)). This regulatory circuit was first discovered in the marine bacterium Vibrio fischeri (Dunlap et al., J. Bacteriol. 164:45-50 (1985)), but later found in P. aeruginosa and many other bacteria (Hastings et al., J. Bacteriol. 181:2667-2668 (1999); de Kievit et al., Infect. Immun. 68:4839-5862 (2000)). The QS system generally consists of two families of proteins, R (regulator) and I (autoinducer synthase) proteins. The R proteins are known to activate transcription of both R and I genes that encode these protein families, as well as numerous downstream targets that include virulence factor and biofilm genes. In contrast, the I proteins are synthases of autoinducer signal molecules (vide infra). Importantly, the R protein is “activated” for DNA binding and transcriptional activation only upon binding to its cognate autoinducer (Gambello et al., J. Bacteriol. 173:3000-3009 (1991)), which is synthesized by the respective I protein. Therefore, as more autoinducers are produced by the I proteins, more activated R proteins exist in the system, which results in increased I protein levels. Thus, it is no coincidence that the signaling molecules are called autoinducers, because of the feedback role they play in QS. In P. aeruginosa, two R proteins, known as LasR and RhlR, and two I proteins, known as LasI and RhlI, have been identified (Passador et al., Science 260:1127-1130 (1993); Gambello et al., J. Bacteriol. 173:3000-3009 (1991); Ochsner et al., J. Bacteriol. 176:2044-2054 (1994); Ochsner et al., Proc. Natl Acad. Sci. USA 92:6424-6428 (1995)). Two autoinducers that bind to the respective R proteins have been identified, and are known as N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) (Pearson et al., Proc. Natl Acad. Sci. USA 91:197-201 (1994); Seed et al., J. Bacteriol. 177:654-659 (1995)) and N-butyloyl-L-homoserine lactone (C4-HSL) (Pearson et al., Proc. Natl Acad. Sci. USA 92:1490-1494 (1995); Winson et al., Proc. Natl Acad. Sci. USA 92:9427-9431 (1995)). For simplicity, hereafter 3-oxo-C12-HSL and C4-HSL are called AI1 and AI2, respectively. By means of these diffusible autoinducers, the bacterial cells can monitor cell density in their immediate surroundings, and regulate gene expression accordingly.
While the las and rhl QS system consists of two separate regulons, their functions are apparently not independent. Even though both AI1-LasR and AI2-RhlR can activate transcription of lasB (which encodes the elastase (LasB) virulence factor), it has been shown that the las QS system controls the rhl QS system at both the transcriptional (Pearson et al., J. Bacteriol. 179:5756-5767 (1997)) and post-translational (Pesci et al., J. Bacteriol. 179:3127-3132 (1997)) levels. For example, as cell density increases, AI1 builds to a critical concentration, at which point it interacts with LasR (Passador et al., Science 260:1127-1130 (1993)). This AI1-LasR complex then activates transcription of a number of genes, such as lasB, toxA, rhlR, and lasI (Whiteley et al., Proc. Natl Acad. Sci. USA 96:13904-13909 (1999); Gambello et al., J. Bacteriol. 173:3000-3009 (1991); Pesci et al., J. Bacteriol. 179:3127-3132 (1997); Storey et al., Infect. Immun. 66:2521-2528 (1998); Toder et al., Infect. Immun. 62:1320-1327 (1994); Gambello et al., Infect. Immun. 61:1320-1327 (1993)). This indicates the two systems are arranged in a hierarchy where the las QS system is dominant over the rhl QS system. Since activation of the lasI gene by the AI1-LasR complex essentially triggers the initial steps in the cascade, LasR is considered the master regulator of P. aeruginosa QS.
More recently, a third signal molecule, 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal, PQS), has been found to provide a possible additional link between the las and rhl QS networks (Pesci et al., Proc. Natl Acad. Sci. USA 96:11229-11234 (1999); McKnight et al., J. Bacteriol. 182:2702-2708 (2000)). These studies showed that PQS strongly induces rhlI while it has lesser positive effects on the transcription of lasR and rhlR. Still, the mechanism by which PQS influences the las and rhl QS networks and the proteins involved in PQS biosynthesis are unknown.
The QS system has been shown to directly relate to bacterial pathogenicity. In particular, a number of reports have shown a positive correlation between QS and P. aeruginosa virulence (Costerton et al., Science 284:1318-1322 (1999); de Kievit et al., Sci. Med. November/December:42-50 (1999); Passador et al., Science 260:1127-1130 (1993); de Kievit et al., Infect. Immun. 68:4839-5862 (2000); Storey et al., Infect. Immun. 66:2521-2528 (1998); Hassett et al., Mol. Microbiol. 34:1082-1093 (1999); Pearson et al., Infect. Immun. 68:4331-4334 (2000); Singh et al., Nature 407:762-764 (2000); Rumbaugh et al., Infect. Immun. 67:5854-5862 (1999); Tang et al., Infect. Immun. 64:37-43 (1996); Davies et al., Science 280:295-298 (1998); Tan et al., Proc. Natl Acad. Sci. USA 96:715-720 (1998); Tan et al., Proc. Natl Acad. Sci. USA 96:2408-2413 (1999); Sawa et al., Infect. Immun. 66:3242-3249 (1998); Telford et al., Infect. Immun. 66:36-42 (1998); Saleh et al., Infect. Immun. 67:5076-5082 (1999); Wu et al., Microbiol. 147:1105-1113 (2001); Silo-Suh et al., Proc. Natl Acad. Sci. USA 99:15699-15704 (2002); Lesprit et al., American Journal of Respiratory and Critical Care Medicine epub (2003)), for review see (Rumbaugh et al., Microbes and Infection 2:1721-1731 (2000)). Disruption of QS genes leads to decreased virulence in mouse, plant, nematode, Drosophila, and wax moth pathogenicity studies. For example, Hamood et al. have shown that the in vivo virulence of lasI, lasR, and rhlI mutants in a burned-mouse model (a commonly used mammalian pathogenicity model) was significantly reduced compared to the wild-type strain with an intact QS cascade (Rumbaugh et al., Infect. Immun. 67:5854-5862 (1999)). While the wild-type strain killed 94% of infected mice, the lasR mutant was able to kill only 28%, and the lasIrhlI double mutant killed only 7%. Expression in trans of constitutively active lasI and rhlI genes in the lasIrhlI mutant restored the ability of the organism to (i) spread within the burned tissue and systemically, and (ii) cause death at near wild-type levels (93%). Ausubel et al. have utilized a C. elegans-P. aeruginosa pathogenesis system to identify virulence-associated genes, and demonstrated that a lasR mutant is significantly less virulent in this surrogate model system (Tan et al., Proc. Natl Acad. Sci. USA 96:715-720 (1998); Tan et al., Proc. Natl Acad. Sci. USA 96:2408-2413 (1999); Mahajan-Miklos et al., Cell 96:47-56 (2003)). Interestingly, the QS mutants behave similarly to mutants with gene disruptions in crucial virulence factor genes. This is the expected result if indeed QS controls expression of virulence factors required for the disease process. It is interesting to note that interference with PQS production led to decreased expression of the LasB elastase virulence factor, further evidence that disruption of the QS system leads to decreased virulence (Calfee et al., Proc. Natl Acad. Sci. USA 98:11633-11637 (2001)).
Another critical discovery was the overlapping role of virulence factors that spanned different phyla (Rahme et al., Science 268:1899-1902 (1995); Tan et al., Proc. Natl Acad. Sci. USA 96:2408-2413 (1999); Mahajan-Miklos et al., Cell 96:47-56 (2003); Rahme et al., Proc. Natl Acad. Sci. USA 94:13245-13250 (1997); Plotnikova et al., Plant Physiol. 124:1766-1774 (2000)). The same P. aeruginosa mutants with reduced virulence in the worm, C. elegans, were also less virulent in the plant and burned mouse models. Based upon such experiments, it was postulated that the bacteria use the same virulence factors for infection of all hosts, a mode dependent upon QS.
A likely explanation for the decrease in virulence when QS is disrupted is that the QS transcriptional activators, LasR and RhlR, directly activate expression of genes encoding critical enzymes (LasB elastase, LasA protease, exoenzyme S, alkaline protease, and phospholipase C) and toxins (rhamnolipid, exotoxin A, cyanide, and pyocyanin) that contribute to the disease process (Rumbaugh et al., Microbes and Infection 2:1721-1731 (2000); de Kievit et al., Sci. Med. November/December:42-50 (1999); Whiteley et al., Proc. Natl Acad. Sci. USA 96:13904-13909 (1999)). QS also regulates components of the MexAB-OprM multidrug efflux pump (Evans et al., J. Bacteriol. 180:5443-5447 (1998)) and genes involved in resistance to reactive oxygen intermediates (sodA, sodB, and katA) (Hassett et al., Mol. Microbiol. 34:1082-1093 (1999)). Interestingly, the ability of P. aeruginosa to form complex, highly developed biofilms is also controlled by QS. Strains unable to form biofilms are less infectious. Davies et al. showed that a lasI mutant produces abnormal (thin and much more uniform) biofilms that are not resistant to detergent treatment. However, in the presence of exogenous AI1, this mutant forms normal detergent-resistant biofilms of an average thickness and cell density similar to that of the wild-type biofilms (Davies et al., Science 280:295-298 (1998)). Singh et al. recently demonstrated that P. aeruginosa in sputum of CF patients are living in the biofilm mode of growth. A direct correlation was made that revealed in vitro bio films and CF sputum harbored 3 times the AI2 relative to AI1 levels (Singh et al., Nature 407:762-764 (2000)), implicating the rhl system in playing a critical role in the biofilm mode of growth. Wu et al. confirmed these results, showing that acyl-HSL molecules are not only expressed by P. aeruginosa infecting mouse lung tissue, but their presence in the lung coincides with the occurrence of pathological changes (Wu et al., Microbiol. 146:2481-2493 (2000)). This strongly suggests that biofilms contribute to the chronic and refractory nature of P. aeruginosa infections in CF lungs, and biofilm formation is dependent on QS. Of particular importance is a study of sputa from lungs of CF patients by Storey et al. which found a direct correlation between the level of lasR mRNA and the level of LasB elastase, LasA protease, exotoxin A, and possibly algD (a gene involved in alginate synthesis, a major component of the biofilm matrix) mRNA (Storey et al., Infect. Immun. 66:2521-2528 (1998)). These studies provide the first direct evidence that QS is indeed active in human lung infections. However, drugs designed to fight P. aeruginosa and QS pathogens based on the QS pathway have not been identified.
In the last few years, investigations of synthetic autoinducer analogs, with respect to agonist and antagonist activities in the QS systems of P. aeruginosa (Passador et al., J. Bacteriol. 178:5995-6000 (1996); Kline et al., Bioorg. Med. Chem. Lett. 9:3447-3452 (1999)), V. fischeri (Schaefer et al., J. Bacteriol. 178:2897-2901 (1996)), and A. tumefaciens (Zhu et al., J. Bacteriol. 180:5398-5405 (1998)), have been reported. In these studies, major efforts were made to vary the length of the aliphatic side chain or modify the 3-oxo functionality of the autoinducers (substituting the 3-oxo group with 3-hydroxyl or methylene groups). The analog molecules whose structures are closely related to the cognate autoinducer structure exhibited weak-to-moderate gene activation (agonist activity). In some cases, the analogs weakly inhibited the induction of target genes (antagonist activity). These studies, however, failed to identify potent synthetic antagonists.
Interestingly, secondary metabolites isolated from the Australian macroalga, Delisea pulchra, exhibited moderate antagonist activity against QS-controlled gene expression in Serratia liquefaciens, V. fischeri, and Erwinia carotovora, all gram-negative bacteria with QS systems similar to that of P. aeruginosa (Givskov et al., J. Bacteriol. 178:6618-6622 (1996); Manefield et al., Microbiol. 145:283-291 (1999); Rasmussen et al., Microbiol. 146:3237-3244 (2000); Manefield et al., FEMS Microbiol. Lett. 205:131-138 (2001)). These natural products are highly halogenated furanone derivatives that are able to act as an antagonist of an AI2-like autoinducer in S. liquefaciens and 3-oxo-C6-HSL in both V. fischeri and E. carotovora. Although their structure, in terms of the electronic and steric features, significantly differs from that of the HSL autoinducers, it still shares the features of the aliphatic side chain and the five-membered lactone ring (but fully conjugated) with the HSL autoinducers. The above naturally occurring furanone was nearly inactive in wild type of P. aeruginosa, PAO1. In 2002, Hentzer et al. reported that a furanone that lacks the alkyl side chain of the natural product exhibits antagonist activity using a PAO-JP2 mini-Tn5-PlasB-gfp(ASV) reporter system (Hentzer et al., Microbiol. 148:87-102 (2002)). Approximately 50% inhibition was observed in the presence of 320 μM of the furanone competing with 0.1 μM AI1. It should be noted that Whiteley et al. grouped QS promoters into four classes based on their responsiveness to AI1 vs. AI2, and the degree and timing of that response (Whiteley et al., Proc. Natl Acad. Sci. USA 96:13904-13909 (1999)). The lasB promoter used in the Hentzer's study is a Class IV promoter that requires both AI1 and AI2 for full induction, and responds very little to AI1 alone. Additionally, they used a low AI1 concentration.
The present invention is directed to overcoming these and other deficiencies in the art.